Biologically Active Natural Products from Fungi: Templates for

14 Biologically Active Natural Products from Fungi: Templates for Tomorrow's Pesticides

Downloaded by EAST CAROLINA UNIV on January 3, 2018 | http://pubs.acs.org Publication Date: July 6, 1984 | doi: 10.1021/bk-1984-0257.ch014

H O R A C E G. C U T L E R Plant Physiology Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Athens, G A 30613

Fungi are a source of unique bioregulators that exhibit a wide range of phytotoxic, or regulatory properties, including fungistatic and bacteriostatic responses. These metabolites o f f e r i n t e r e s t i n g templates for p o t e n t i a l agricultural use. Minor changes in molecules enhance specific activity. A sensitive, relatively simple, primary plant bioassay is used to d e t e c t biological activity and novel structures including pergillin, dihydropergillin, chaetoglobosin K, h y d r o x y t e r p h e n y l l i n and orlandin have been isolated using this assay. Other metabolites have also shown selective activity against higher plants; cytochalasin H controls flowering in tobacco plants; cladosporin diacetate induces chlorosis in corn p l a n t s ; prehelminthosporol causes necrosis and stunting in corn. Terphenyllin and 6-pentyl-α-pyrone have either f u n g i s t a t i c , or b a c t e r i o s t a t i c properties. Biodegradable p r o p e r t i e s , high specificity and low dosage rates make fungal metabolites, and d e r i v a t i v e s , attractive candidates for agricultural use. Biologically a c t i v e n a t u r a l products from fungi, the so-called secondary metabolites, are of primary importance as a source of novel compounds which may be used to c o n t r o l p l a n t growth and development. These compounds tend to be s p e c i f i c in t h e i r a c t i o n on t a r g e t p l a n t s , are generally e f f e c t i v e at low concentrations, and tend to be non-persistent in the environment. Furthermore, the compounds o f f e r unique templates f o r f u r t h e r synthesis of analogs or a d d i t i o n a l syntheses that s t a r t with f e r m e n t a t i o n products. E f f e c t s o f f u n g a l l y d e r i v e d n a t u r a l products on p l a n t s include such phenomena as i n h i b i t i o n of v e g e t a t i v e growth, shoot This chapter not subject to U.S. copyright. Published 1984, American Chemical Society

Ory and Rittig; Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1984.


154

BIOREGULATORS: CHEMISTRY AND USES

s u p p r e s s i o n , or i n h i b i t i o n of f l o w e r i n g , depending upon the species treated. Some effects may be c l a s s i f i e d as t r u l y growth r e g u l a t o r y , o t h e r s as p h y t o t o x i c . Minor changes in an active molecule may i n c r e a s e the s p e c i f i c a c t i v i t y so as to i n d u c e c h l o r o s i s in s e l e c t p l a n t s or change morphological development. I n h i b i t i o n of growth is not only limited to higher plants but may a l s o i n c l u d e the f u n g i and b a c t e r i a . The l a t t e r , of course, is well documented in the study of pharmaceuticals. In order to d e t e c t b i o l o g i c a l l y active natural products in crude extracts of fungi and to s u c c e s s f u l l y i s o l a t e the a c t i v e p r i n c i p l e in pure form, i t is necessary to have a highly r e l i a b l e bioassay. The assay s h o u l d be b r o a d in r e s p o n s e , ideally d e t e c t i n g a l l m a n i f e s t a t i o n s of growth i n h i b i t i o n , or growth promotion (that is, i t should c l e a r l y e x h i b i t p h y t o t o x i c i t y , f u n g i s t a t i c or b a c t e r i o s t a t i c properties, i n h i b i t i o n of c e l l wall extension and m i t o s i s , and stem c u r v a t u r e ) . F u r t h e r , the assay should be r e l a t i v e l y simple, highly r e l i a b l e , quick, and r e s u l t s should be c r i t i c a l l y r e p r o d u c i b l e from assay to assay. These c h a r a c t e r i s t i c s are important because often the chemistry may be apparently anomalous d u r i n g the i s o l a t i o n of n a t u r a l p r o d u c t s . For example, a major m e t a b o l i t e may appear as a single spot on thin-layer plates when developed and observed under u l t r a - v i o l e t l i g h t or t r e a t e d w i t h a chromogenic agent. But often, the major v i s i b l e m e t a b o l i t e is not the b i o l o g i c a l l y a c t i v e m a t e r i a l . I n s t e a d , the active metabolite may be i n v i s i b l e , or may appear as a very minor spot. The former e s p e c i a l l y occurs with compounds t h a t have high s p e c i f i c a c t i v i t y ; and there are always those metabolites that do not have conjugated double bonds, or s u i t a b l e c h r o m o p h o r e s , or a r e h i d d e n under b i o l o g i c a l l y i n a c t i v e metabolites even in d i f f e r e n t developing solvents. Consequently, the primary b i o a s s a y is of paramount importance and must be an unerring source of i n f o r m a t i o n concerning the whereabouts and n a t u r e o f the m e t a b o l i t e being t r a c k e d d u r i n g an i s o l a t i o n exercise. Because of the importance of the b i o a s s a y the technique, which has been published elsewhere (1.) is nevertheless presented again with some f u r t h e r t e c h n i c a l d e t a i l s and discussion. The b i o a s s a y is b a s e d on an e a r l i e r method (2) which has been m o d i f i e d . Wheat seed (Triticum aestivum L. cv Wakeland) are sown on moist sand in p l a s t i c trays, covered with aluminum f o i l , placed in the dark at 22±1°C. f o r 4 days, then the etiolated plants are harvested. E n t i r e shoots are cut from the seed and r o o t s and p l a c e d , apex f i r s t , into a Van der Weij g u i l l o t i n e . The a p i c a l 2 mm are discarded and the next 4 mm are cut and placed in d i s t i l l e d water in a p e t r i d i s h and soaked p r i o r to use. Generally, compounds to be tested for a c t i v i t y , either in crude e x t r a c t s or pure, are d i s s o l v e d in a suitable solvent and a known volume (or concentration) is placed in a t e s t tube and then evaporated to dryness under a stream of n i t r o g e n . When pure compounds are t e s t e d they are u s u a l l y d i s s o l v e d in a c e t o n e up t o 7.5 uL a c e t o n e / l mL of f i n a l solution. Previous studies have shown that t h i s quantity of acetone is not p h y t o t o x i c to e i t h e r oat f i r s t internode segments (3.) or wheat c o l e o p t i l e s (.4) Then 2 mL of p h o s p h a t e - c i t r a t e b u f f e r (pH 5.6) containing 2% sucrose (5.) are



14.

CUTLER

Templates for Tomorrow's Pesticides

155

added to the t e s t tube c o n t a i n i n g the test material and control tubes are prepared with buffer and sucrose only. Next, ten washed c o l e o p t i l e segments are added to each test tube and the tubes are placed in an i n c l i n e d r o l l e r - t u b e apparatus that rotates 1/5 rpm. A l l procedures are carried out under a green s a f e l i g h t at 540 nm, a photomorphogenetically inactive wavelength. A f t e r 18-24 hours at 22°C. (the time does not have to be exact from t e s t to test because a l l f i n a l measurements are r e l a t i v e to c o n t r o l s e c t i o n s w i t h i n a s p e c i f i c assay) the sections are removed from the tubes, blotted dry on paper towels, placed on a glass p l a t e , put i n t o a photographic e n l a r g e r to p r o j e c t a X3 image and measured (6). D a t a a r e s u b j e c t e d t o s t a t i s t i c a l a n a l y s i s by a m u l t i p l e comparison procedure (7). While the mechanics of the bioassay are straightforward there are subtle points that need to be emphasized. F i r s t , the sections cut in the g u i l l o t i n e a r e very a c c u r a t e . Second, c o l e o p t i l e segments a r e hollow c y l i n d e r s of t i s s u e with c u t e n d s . The s u r f a c e area that comes i n t o contact with the test solution is larger than i t would be with a s o l i d cylinder of t i s s u e , asisthe case with oat mesocotyls. Third, because the segments are rotated they are constantly bathed and agitated during the course of t h e experiment. And f o u r t h , and most important, the success of the bioassay depends on the genotype of the wheat used. T h i s f i n a l p o i n t cannot be over s t r e s s e d and a d i l i g e n t e f f o r t to find and choose a genotype for the type of study at hand is e s s e n t i a l . The t e s t of any bioassay as a r e s e a r c h t o o l is d i r e c t l y proportional to the v a l i d i t y of the r e s u l t s o b t a i n e d . Using the c o l e o p t i l e b i o a s s a y as the primary t e s t , we now discuss some of the novel metabolites that have been i s o l a t e d from f u n g i which e l i c i t s p e c i f i c responses in higher plants, or fungi, or bacteria and which may e v e n t u a l l y f i n d use as s e l e c t i v e h e r b i c i d e s , anti-flowering agents, fungicides and bactericides in a g r i c u l t u r a l management. While fungal m e t a b o l i t e s that e x h i b i t p h y t o t o x i c , plant growth i n h i b i t o r y and other properties have been isolated by other researchers, t h i s discussion is l i m i t e d to those compounds that we have detected with t h i s bioassay. P e r g i l l i n and d i h y d r o p e r g i l l i n ( F i g u r e s 1 and 2), were two m e t a b o l i t e s i s o l a t e d from c u l t u r e s of A s p e r g i l l u s ustus (ATCC 38849) (8.9). This genus and species, though not necessarily t h i s a c c e s s i o n , have y i e l d e d k o j i c acid (10) austdiol (11) and austin (12) which a r e a l l v e r t e b r a t e t o x i n s . Crude e x t r a c t s of the f u n g u s i n h i b i t e d t h e growth of wheat c o l e o p t i l e s ; and by a succession of séparatory techniques p e r g i l l i n and d i h y d r o p e r g i l l i n were e v e n t u a l l y i s o l a t e d , though not at the same time. As the authors stated in their i s o l a t i o n of d i h y d r o p e r g i l l i n , since the wheat c o l e o p t i l e cannot d i s c r i m i n a t e between i n d i v i d u a l i n h i b i t o r s , on a gross s c a l e , i t was i m p o s s i b l e to determine whether a c t i v i t y in tubes 31-51 was attributable to d i l u t e amounts of p e r g i l l i n ( s p e c i f i c a c t i v i t y data were u n a v a i l a b l e at that time) or another i n h i b i t o r . I t is now obvious that tubes 31-51 contained d i h y d r o p e r g i l l i n . Furthermore, after the c r y s t a l l i z a t i o n o f p e r g i l l i n from tubes 52-58, there were recoverable traces of d i h y d r o p e r g i l l i n in the supernatant l i q u i d . " (9.) . T h i s statement shows the s t r e n g t h and weakness of the




Figure 2

Dihydropergillin



14.

CUTLER

Templates for Tomorrow's Pesticides

157

bioassay system, especially when two active metabolites overlap as they exit a column upon chromatography. Upon e x a m i n i n g t h e m o l e c u l e s o f p e r g i l l i n and d i h y d r o p e r g i l l i n i t will be noted that the only difference is that in the l a t t e r the C12-13 bond is s a t u r a t e d . T h i s allows the isopropyl group to form a staggered conformation with the fur an r i n g and a c c o r d i n g l y a l t e r s the b i o l o g i c a l a c t i v i t y . Thus, e t i o l a t e d c o l e o p t i l e s are inhibited 50% at 10~,M with p e r g i l l i n , while d i h y d r o p e r g i l l i n i n h i b i t s 100% (Figures 3 and 4). At 10~,M d i h y d r o p e r g i l l i n s i g n i f i c a n t l y i n h i b i t s 7% more than p e r g i l l i n . Minor changes in a m o l e c u l e may be r e a d i l y d e t e c t e d by the bioassay and further evidence for t h i s will be presented. Chaetoglobosin K, was one of the more d i f f i c u l t metabolites to i s o l a t e and was o b t a i n e d from Diplodia macrospora (ATCC 36896) ( 13) ( F i g u r e 5 ) . I n i t i a l e x t r a c t i o n s of the mycelium were made w i t h a c e t o n e ; and when a l i q u o t s o f the crude e x t r a c t were b i o a s s a y e d , they i n h i b i t e d c o l e o p t i l e s some 80% r e l a t i v e t o c o n t r o l s . While such a f i g u r e is s t a t i s t i c a l l y s i g n i f i c a n t , we have generally found that t h i s l e v e l of a c t i v i t y does not warrant f u r t h e r i s o l a t i o n e f f o r t . As a r u l e , a c t i v i t y decreases with purity (for example, in c o n s t r a s t to the enrichment process in enzyme p u r i f i c a t i o n . ) The reason appears t o be that crude p r e p a r a t i o n s c o n t a i n c o - f a c t o r s or r e l a t e d compounds t h a t s y n e r g i z e . However, in this case p u r i f i c a t i o n progressed to the point that c r y s t a l s were obtained with great d i f f i c u l t y . And, e x c e p t i o n a l l y , the s p e c i f i c a c t i v i t y i n c r e a s e d with p u r i t y . Chaetoglobosin K, is active at 10~?M in the bioassay (Figure 6). The molecule is unique in that u n l i k e o t h e r members o f t h e chaetoglobosin family, there is an additional methyl group at CIO and C l l (1Λ). Subsequent work (15) has disclosed another member of the f a m i l y with the same s u b s t i t u e n t s which lacks the C6-C7 epoxide, named chaetoglobosin L. It is especially i n t e r e s t i n g to n o t e t h a t c h a e t o g l o b o s i n Κ may be c o n s i d e r e d an i n d o l e - 3 - y l compound and those compounds have h i s t o r i c a l l y been promoters of wheat c o l e o p t i l e s (,16) . O b v i o u s l y , c h a e t o g l o b o s i n Κ is exceptional in t h i s regard. Yahara (17) has tested chaetoglobosin Κ and o t h e r cy t ο cha l a s i n s and c h a e t o g l o b o s i n s in C3H mouse f i b r o p l a s t i c c e l l s . Chaetoglobosin K, at 20 uM, caused c u r v a t u r e of a c t i n cables, normally present in a straight configuration; but the same concentration did not induce rounding up of f i b r o p l a s t s . At O.2 uM membrane r u f f l i n g , a function of c e l l locomotion, was strongly i n h i b i t e d . Because of very l i m i t e d q u a n t i t i e s , t h e metabolite could not be tested in other b i o l o g i c a l systems. H y d r o x v t e r p h e n v l l i n . ( 2 ' , 5 ' -dimethoxy -3,4,3',4" -tetrahydroxy-p-terphenyl) was a curious metabolite that exhibited b i o l o g i c a l a c t i v i t y in the primary bioassay (18) (Figure 7). I t was obtained from cultures of A s p e r g i l l u s candidus (ATCC 36008). A d i f f e r e n t s t r a i n of this organism has yielded k o j i c acid (10). The compound is c u r i o u s because, on viewing the structure, the f i r s t o b s e r v a t i o n is that the m e t a b o l i t e is not a ' t y p i c a l ' n a t u r a l product and that i t best represents a synthetic product because of the c h a r a c t e r i s t i c carbon to carbon bonding between the three r i n g s . Fused r i n g s t r u c t u r e s are commonly found natural products and modified biphenyl compounds have been isoalted (vide




158

Figure 3

I n h i b i t o r y e f f e c t of p e r g i l l i n on the growth of wheat c o l e o p t i l e s (T. aestivum L. » cv. Wake land). Control: dotted l i n e . Significant inhibition: below s o l i d l i n e (P

Biologically Active Natural Products from Fungi: Templates for

Recommend Documents